There is considerable evidence now available that supports an important rote for the serotonin (5-HT) transporter, a member of the NaCl- dependent transporter gene family, in the development of pulmonary vascular remodeling and hypertension. Transcription of this transporter is stimulated by hypoxia and the transporter participates in 5-HT-induced pulmonary artery (PA) smooth muscle cell (SMC) hyperplasia/hypertrophy. Based on our studies done largely during the last funding period, we hypothesize that there are specific intermediate intracellular signaling pathways through which the mitogenic action of 5-HT occurs (denoted in Figure 1). We plan in this proposal to further study these intermediate signals by: 1) examining the PA SMC NAD(P)H oxidase that is stimulated in response to 5-HT transport; 2) characterizing the role of Src-Fak non-receptor kinases in signal transduction, cyclin D1 gene regulation, cell growth and cytoskeletal reorganization produced by 5-HT; 3) evaluating transcription factors (in particular STAT and GATA) that may be linked to 5-HT transporter stimulation of SMC growth; and 4) assessing the role of a concomitant 5-HT4-like receptor action on the same SMC that can convert the growth stimulatory effect to a growth inhibitory one. For these studies we will use bovine PA SMCs, with which we have had considerable experience, but will shift to PA SMCs of the human, rat and mouse species as needed to answer specific questions, or where reagents being used require specific species homology. Methodologies will include, but not be limited to, Western and Northern analyses, electrophoretic mobility shift assays, immunoblotting, PCR technology, lipid-mediated transient transfections, adenoviralmediated gene transfer, and use of reporter gene constructs, all of which are ongoing in our laboratory. We anticipate that a fuller understanding of the intracellular processes responding to 5-HT transporter activation may allow development of new strategies for treating pulmonary hypertension.